WO2021092689A1 - Procédés de culture de micro-organismes - Google Patents

Procédés de culture de micro-organismes Download PDF

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WO2021092689A1
WO2021092689A1 PCT/CA2020/051537 CA2020051537W WO2021092689A1 WO 2021092689 A1 WO2021092689 A1 WO 2021092689A1 CA 2020051537 W CA2020051537 W CA 2020051537W WO 2021092689 A1 WO2021092689 A1 WO 2021092689A1
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light
acid
light spectrum
period
photosynthetic microorganism
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Nikunj SHARMA
Isabel DESGAGNÉ-PENIX
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Algae-C Inc.
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    • C12Y—ENZYMES
    • C12Y404/00—Carbon-sulfur lyases (4.4)
    • C12Y404/01—Carbon-sulfur lyases (4.4.1)
    • C12Y404/01026—Olivetolic acid cyclase (4.4.1.26)
    • C—CHEMISTRY; METALLURGY
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
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    • C12Y121/00—Oxidoreductases acting on X-H and Y-H to form an X-Y bond (1.21)
    • C12Y121/03—Oxidoreductases acting on X-H and Y-H to form an X-Y bond (1.21) with oxygen as acceptor (1.21.3)
    • C12Y121/03007—Tetrahydrocannabinolic acid synthase (1.21.3.7)
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    • C12Y—ENZYMES
    • C12Y121/00—Oxidoreductases acting on X-H and Y-H to form an X-Y bond (1.21)
    • C12Y121/03—Oxidoreductases acting on X-H and Y-H to form an X-Y bond (1.21) with oxygen as acceptor (1.21.3)
    • C12Y121/03008—Cannabidiolic acid synthase (1.21.3.8)
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    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)

Definitions

  • the present disclosure relates to methods of culturing microorganisms or genetically engineered microorganisms which produce at least one cannabinoid biosynthetic pathway product.
  • PNPs valuable plant natural products
  • the commercialization of valuable plant natural products (PNPs) is often limited by the availability of PNP producing-plants, by the low accumulation of PNPs in planta and/or the time-consuming and often inefficient extraction methods not always economically viable.
  • PNPs of commercial interest is often challenging.
  • the recent progress in genetic engineering and synthetic biology makes it possible to produce heterologous PNPs or other biosynthetic products in microorganisms such as bacteria, yeast and microalgae.
  • engineered microorganisms have been reported to produce the antimalarial drug artemisinin and of the opiate (morphine, codeine) painkiller precursor reticuline (Keasling 2012; Fossati et al 2014; DeLoache et al 2015).
  • opiate morphine, codeine
  • reticuline reticuline
  • the latest metabolic reactions to yield the valuable end-products such as codeine and morphine in genetically modified yeast-producing reticuline have yet to be successfully achieved.
  • bacterial or yeast platforms do not support the assembly of complex PNP or biosynthetic product pathways.
  • microalgal cells have been suggested to possess advantages over other microorganisms, including the likelihood to perform similar post-translational modifications of proteins as plant and recombinant protein expression through the nuclear, mitochondrial or chloroplastic genomes (Singh et al 2009).
  • Cannabinoid biosynthetic pathway products such as D9- tetrahydrocanannabinol and other cannabinoids (CBs) are polyketides responsible for the psychoactive and medicinal properties of Cannabis sativa. More than 110 CBs have been identified so far and are all derived from fatty acid and terpenoid precursors (ElSohly and
  • the first metabolite intermediate in the CB biosynthetic pathway in Cannabis sativa is olivetolic acid that forms the polyketide skeleton of cannabinoids.
  • a type III polyketide synthase (PKS; also known as tetraketide synthase (TKS) orolivetol synthase) enzyme condenses hexanoyl-CoA with three malonyl-CoA in a multi-step reaction to form trioxododecanoyl-CoA.
  • OAC olivetolic acid cyclase
  • CB diversification is generated by the sequential action of “decorating” enzymes on the OA backbone.
  • the gene sequence for PKS and OAC have been identified and characterized in vitro (Lussier2012; Gagne ef a/2012; Marks et al 2009; Stout et a/ 2012; Taura et al 2009).
  • Microorganisms such as microalgae and cyanobacteria
  • Light is the energy source harvested by the photosynthetic machinery of autotrophic (such as photoautotrophic or photosynthetic) microorganisms.
  • autotrophic such as photoautotrophic or photosynthetic
  • microalgae can adapt to variations in light spectrum and intensity. It has been shown that light quality has an impact on chloroplast migration, zygote germination, and light acclimation (Furukawa et al 1998; Shikata et al 2011 ; Holdsworth et al 1985).
  • Recent discoveries have revealed photoreceptors such as red light sensing phytochromes, blue- light sensing cryptochromes and aureochromes (Armbrust et al 2004; Bowler et al 2008).
  • the present disclosure describes a method for culturing a microorganism under a first light spectrum for a first period of time and subsequently in a second light spectrum for a second period of time.
  • the present disclosure provides a method of culturing a photosynthetic microorganism, comprising: a. culturing the photosynthetic microorganism under a first light spectrum for a first period of time; and b. culturing the photosynthetic microorganism under a second light spectrum for a second period of time, wherein the second light spectrum is different from the first light spectrum.
  • the present disclosure further provides a method of culturing a photosynthetic microorganism capable of producing at least one biosynthetic product in a culture medium, comprising (a) culturing the microorganism under a first light spectrum for a first period of time; and (b) culturing the microorganism under a second light spectrum for a second period of time, wherein the second light spectrum is different from the first light spectrum, and wherein the culture medium comprises a 2,4-dihydroxy-6- alkylbenzoic acid or a 2,4-dihydroxy-6-alkylbenzoate.
  • Figure 1 An exemplary cannabinoid biosynthetic pathway based on enzymes from Cannabis sativa
  • FIG. 1 Gravimetric biomass analysis in mixotrophic and autotrophic conditions under different colours of light (White Light [C], Red Light [R], and Yellow Light [Y]).
  • C, R, and Y refer to autotrophic cultures;
  • C1 , R1 , and Y1 refers to mixotrophic cultures in L1 media supplemented with 1 % glucose;
  • C2, R2, and Y2 refers to mixotrophic cultures in L1 media supplemented with 1 % Glycine.
  • FIG. 3 Gravimetric total lipid analysis in mixotrophic and autotrophic conditions under different colours of light (White Light [C], Red Light [R] and Yellow Light [Y]).
  • C, R, and Y refer to autotrophic cultures;
  • C1 , R1 , and Y1 refers to mixotrophic cultures in L1 media supplemented with 1 % glucose;
  • C2, R2, and Y2 refers to mixotrophic cultures in L1 media supplemented with 1 % Glycine.
  • Statistical analysis by one-way ANOVA indicated statistical significance for pairings C1 vs R2, R1 vs R2, R2 vs Y, and R2 vs Y1.
  • Figure 4 Cell growth analysis of P.tricornutum measured by optical density at 680 nm at different days during autotrophic culture.
  • Absorbance units (a.u.) are reported.
  • FIG. 5 Growth curve of P.tricornutum in autotrophic culture in different light conditions as measured by optical density at 680 nm: white light (C); red light (R), red light during lag and exponential phases (Rs); red light during lag phase (RT1); red light during exponential phase (RT2); red light during stationary phase (RT3). Absorbance units (a.u.) are reported.
  • FIG. Biomass analysis of autotrophic cultures under white light (C), red light (R), yellow light (Y), and red light under the lag and exponential phases (RS). Statistical analysis by one-way ANOVA indicated statistical significance for pairing C vs RS.
  • FIG. 7 Total lipid analysis of autotrophic cultures under white light (C), red light (R), yellow light (Y), and red light under the lag and exponential phases (RS). Statistical analysis by one-way ANOVA indicated statistical significance for pairing C vs RS.
  • Figure 8 Visual analysis of the lipid droplets in P.tricornutum grown in white light (left images) and red light (right images). Bright field (top images) and fluorescent (bottom images).
  • Figure 9 Transmittance spectrum of red light colour filter from 400nm to 720nm.
  • Figure 10 Biomass of analysis of autotrophic cultures under white light (C), blue light (B), blue light during lag and exponential phases (Bs), yellow light (Y), yellow light during lag and exponential phases (Ys), red light (r), and red light during lag and exponential phases (rs).
  • the present disclosure provides the discovery that culturing a photosynthetic microorganism under a first light spectrum for a period of time and then subsequently growing said microorganism under second light spectrum may increase biomass and/or lipid production, which may in turn improve the production of a biosynthetic product in said microorganism.
  • the microorganism may produce a cannabinoid biosynthetic pathway product, the production of which is improved by the methods of the present invention.
  • the improvement in the production of at least one cannabinoid biosynthetic pathway product results from increased cell growth, biomass, and/or lipid production of said cultured microorganism cultured using methods provided herein.
  • Increasing biomass and cell growth can increase the total yield of the desired product, and can increase the number of culture cycles in a given period of time.
  • Increasing lipid production, particularly of short fatty acids, such as hexanoic acid and malonyl-CoA may increase precursor substrates for cannabinoid biosynthesis and thus may increase the total yield of desired products.
  • Increasing lipid production may also facilitate the downstream extraction of hydrophobic cannabinoid biosynthetic pathway products from the biomass, and may further improve the viability of microorganisms producing cannabinoid biosynthetic pathway products by allowing the microorganisms to sequester toxic cannabinoid biosynthetic pathway products in intracellular lipid bodies.
  • the present disclosure provides a method of culturing a photosynthetic microorganism, comprising: a. culturing the photosynthetic microorganism under a first light spectrum for a first period of time; and b. culturing the photosynthetic microorganism under a second light spectrum for a second period of time, wherein the second light spectrum is different from the first light spectrum.
  • the present disclosure further provides a method of culturing a photosynthetic microorganism capable of producing at least one biosynthetic product in a culture medium, comprising (a) culturing the microorganism under a first light spectrum for a first period of time; and (b) culturing the microorganism under a second light spectrum for a second period of time, wherein the second light spectrum is different from the first light spectrum, and wherein the culture medium comprises a 2,4-dihydroxy-6- alkylbenzoic acid or a 2,4-dihydroxy-6-alkylbenzoate.
  • Photosynthetic microorganisms are capable of carbon fixation wherein carbon dioxide (which is not a fixed carbon source) is fixed into organic molecules such as sugars using energy from a light source.
  • the fixation of carbon dioxide using energy from a light source is photosynthesis. Suitable sources of light for the provision of energy in photosynthesis include sunlight and artificial lights.
  • Photosynthetic microorganisms are capable of growth and/or metabolism without a fixed carbon source.
  • microalgae can fix carbon dioxide from a variety of sources, including atmospheric carbon dioxide, industrially-discharged carbon dioxide (e.g. flue gas and flaring gas), and from soluble carbonates (e.g.
  • a non-fixed carbon source such as carbon dioxide can be added to a culture of microalgae by injection or by bubbling of a carbon dioxide gas mixture into the culture medium.
  • Photosynthetic growth is a form of autotrophic growth, wherein a microorganism is able to produce organic molecules on its own using an external energy source such as light. This is in contrast to heterotrophic growth, wherein a microorganism must consume reduced organic molecules for growth and/or metabolism. Heterotrophic microorganisms therefore require a fixed carbon source for growth and/or metabolism.
  • Some photosynthetic microorganisms are capable of mixotrophic growth, wherein the microorganism fixes carbon by photosynthesis while also consuming fixed carbon sources.
  • the autotrophic metabolism is integrated with a heterotrophic metabolism that oxidizes reduced carbon sources available in the culture medium.
  • Photosynthetic microalgae are commonly cultivated in mixotrophic conditions by adding fixed carbon sources as described herein to the culture medium.
  • fixed carbon sources include, but are not limited to, sugars (e.g. glucose, galactose, mannose, fructose, sucrose, lactose), amino acids or amino acid derivatives (e.g.
  • microorganisms such as microalgae and cyanobacteria may be cultured using methods and conditions known in the art (see, e.g., Biofuels from Algae, eds. Pandey et al., 2014, Elsevier, ISBN 978-0- 444-59558-4). Some microorganisms are capable of chemoautotrophic growth.
  • chemoautotrophic organisms are capable of carbon dioxide fixation but using energy derived from chemical sources (e.g. hydrogen sulfide, ferrous iron, molecular hydrogen, ammonia) rather than light.
  • energy derived from chemical sources e.g. hydrogen sulfide, ferrous iron, molecular hydrogen, ammonia
  • Photosynthetic culture of a microorganism requires the provision of light.
  • Various sources of light may be used, including solar light and artificial light.
  • Artificial light may be provided from a variety of sources known in the art, including but not limited to, fluorescent lights, high intensity discharge lights, halide lights, and light-emitting diodes (LED).
  • the use of artificial light sources allows for the control of the light spectrum provided to the culture.
  • the light spectrum provided to a culture can also be controlled by passing white light through a colour filter.
  • Light spectrum refers to a defined wavelength, a plurality of defined wavelengths, or range of wavelengths of light. A light spectrum can therefore comprise a single wavelength of light, multiple wavelengths of light, or an entire range of wavelengths of light.
  • Visible light is typically defined in the art as light spectrum comprising about 400 nm to about 700 nm.
  • colour spectra typically defined in the art as violet light (e.g. about 400 nm to about 450 nm), blue light (e.g. about 450 nm to about 485 nm), cyan light (e.g. about 485 nm to about 500 nm), green light (e.g. about 500 nm to about 565 nm), yellow light (e.g.
  • Solar light comprises both white light as well as ultraviolet light (about 10 nm to about 400 nm) and infrared light (about 700 nm to about 1 mm).
  • the first light spectrum comprises at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of light within about 590 nm to about 740 nm, about 625 nm to about 740 nm, about 625 nm to about 720 nm, about 625 nm to about 700 nm, about 600 nm to about 730 nm, about 610 nm to about 720 nm, about 620 nm to about 710 nm, about 630 nm to about 700 nm, about 640 nm to about 690 nm, about 650 nm to about 680 nm, about 660 nm to about 670 nm, about 590 nm to about 625 nm, about 625 nm to about 660 nm, about 660 nm to about 695 nm, about 680 n
  • percent (%) of light it is meant the portion of the light within a defined range of wavelengths as a percentage of the total light emitted.
  • the percent (%) of light may calculated as a percent of total light by units of measuring light known in the art including lux (lx), lumen (Im), pmol nr 2 s 1 , and/or as a percent of the total transmitted light.
  • the first light spectrum comprises about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% of light within about 590 nm to about 740 nm, about 625 nm to about 740 nm, about 625 nm to about 720 nm, about 625 nm to about 700 nm, about 600 nm to about 730 nm, about 610 nm to about 720 nm, about 620 nm to about 710 nm, about 630 nm to about 700 nm, about 640 nm to about 690 nm, about 650 nm to about 680 nm, about 660 nm to about 670 nm, about 590 nm to about 625 nm, about 625 nm to about 660 nm, about 660 nm to about 695 nm, about 680 nm to about 700 nm,
  • the first light spectrum comprises about 65% to about 95%, about 70% to about 95%, about 75% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 65% to about 90%, about 70% to about 90%, about 75% to about 90%, about 80% to about 90%, about 85% to about
  • the second light spectrum comprises or consists essentially of light within about 400 nm to about 700 nm, about 400 nm to about 740 nm, about 400 nm to about 450 nm, about 450 nm to about 485 nm, about 485 nm to about 500 nm, about 500 nm to about 565 nm, about 565 nm to about 590 nm, about 410 nm to about 690 nm, about 420 nm to about 680 nm, about 430 nm to about 670 nm, about 440 nm to about 660 nm, or about 450 nm to about 650 nm.
  • the second light spectrum comprises or consists essentially of solar light.
  • Methods provided herein involve culturing a microorganism under at least two different light spectra.
  • the first light spectrum comprises at least 65% of light within about 590nm to 740nm.
  • Light perceived by red-light receptors (phytochromes) can regulate nutrient metabolism, regulation of cellular events and signalling cascades (Wang et al 2015).
  • the second light spectrum comprises or consists essentially of blue light, white light, or solar light.
  • methods described herein involve withdrawal of the first light spectrum and introduction of the second light spectrum.
  • methods described herein involve maintaining the first light spectrum while introducing a different light spectrum to form the second light spectrum.
  • methods described herein involve adjusting the first light spectrum to form the second light spectrum, for example, by removing light of certain wavelengths within the spectrum and/or adding light within the same spectrum but having different wavelengths.
  • the culture of microorganisms comprises distinct phases. Initially, a new culture of microorganisms exists in a lag phase, wherein the microorganisms are adapting to the culture conditions. In the lag phase, the microorganisms are active but cell division is low or is not occurring. Subsequent to the lag phase, the rate of cell division accelerates as the culture enters the exponential phase, wherein nutrients are typically in excess and the microorganisms multiply exponentially at a constant rate. When a culture of microorganisms reaches a high density, the availability of nutrients begins to decline, and/or metabolic waste products accumulate in the culture environment, causing the culture to enter a stationary phase wherein the rate of cell division begins to decline.
  • the rate of cell division will slow down and the population of the culture reaches a plateau. If a culture is allowed to continue for long enough in the stationary phase then a death phase will begin wherein microorganisms die due to nutrient starvation and/or the toxicity of metabolic waste products, and the population of the culture declines. It is within the ability of the skilled person to determine the phase of a culture of microorganisms by methods known in the art, for example by using optical density to determine the cell density within the culture.
  • Methods provided herein involve changing the first light spectrum to the second light spectrum at a selected time point during a phase or as one phase ends and the next phase starts.
  • the first light spectrum is changed to the second light spectrum at the end of the lag phase, at the beginning of the exponential phase, during the exponential phase, at the end of the exponential phase, or at the beginning of the stationary phase.
  • microorganisms for use in the present invention can be genetically engineered to express one or more proteins or enzymes to produce a biosynthetic product.
  • a “biosynthetic product” is a desired product produced by a cultured microorganism including, but not limited to, a polypeptide, a lipid, a nucleic acid, a polysaccharide, a glycoprotein, or an organic molecule.
  • a biosynthetic product may be produced naturally by a wild-type microorganism in culture, produced by the conversion of a substrate or precursor in the culture medium by a microorganism in culture, or produced by the expression of exogenous genes in a microorganism in culture.
  • the biosynthetic product is a cannabinoid biosynthetic pathway product.
  • the microorganism is engineered to express enzymes of a cannabinoid biosynthetic pathway to produce a cannabinoid biosynthetic pathway product.
  • the term “genetically engineered” refers to the alteration of genetic material of an organism using molecular biology techniques known in the art, including but not limited to, introducing exogenous genes into a microorganism via episomes, integrative vectors, and homologous recombination vectors; DNA editing by zinc fingers or CRISPR-Cas; and/or by transformation methods known in the art.
  • the genetically engineered microorganism includes a living modified microorganism, genetically modified microorganism or a transgenic microorganism. Genetic engineering includes addition, deletion, modification and/or mutation of genetic material.
  • nucleic acid molecule is intended to include unmodified DNA or RNA or modified DNA or RNA.
  • nucleic acid molecules of the disclosure may be composed of single- or double-stranded DNA; DNA that is a mixture of single- and double-stranded regions; single- or double-stranded RNA; RNA that is a mixture of single- and double-stranded regions; hybrid molecules comprising DNA and RNA that may be single-stranded, double-stranded, or a mixture of single- and double-stranded; or circular or linearized DNA-based vectors.
  • the nucleic acid molecules of the disclosure may contain one or more modified bases.
  • Modified bases include, for example, tritiated bases and unusual bases such as inosine.
  • a variety of modifications can be made to DNA and RNA; thus "nucleic acid molecule” embraces chemically, enzymatically, or metabolically modified forms.
  • the term “polynucleotide” shall have a corresponding meaning.
  • the genetically engineered microorganism comprises at least one nucleic acid molecule encoding a polypeptide disclosed herein.
  • exogenous refers to an element that has been introduced into a cell.
  • An exogenous element can include a polypeptide or a nucleic acid.
  • An exogenous nucleic acid is a nucleic acid that has been introduced into a cell, such as by a method of transformation.
  • An exogenous nucleic acid may code for the expression of an RNA and/or a polypeptide.
  • An exogenous nucleic acid may have been derived from the same species (homologous) orfrom a different species (heterologous).
  • An exogenous nucleic acid may comprise a homologous sequence that is altered such that it is introduced into the cell in a form that is not normally found in the cell in nature.
  • an exogenous nucleic acid that is homologous may contain mutations, be operably linked to a different control region, or be integrated into a different region of the genome, relative to the endogenous version of the nucleic acid.
  • An exogenous nucleic acid may be incorporated into the chromosomes of the transformed cell in one or more copies, into the plastid or mitochondrial DNA of the transformed cell, or be maintained as a separate nucleic acid outside of the transformed cell genome.
  • the term “vector” or “nucleic acid vector” means a nucleic acid molecule, such as a plasmid, comprising regulatory elements and a site for introducing transgenic DNA, which is used to introduce said transgenic DNA into a microorganism.
  • the transgenic DNA can encode a heterologous polypeptide, which can be expressed in and isolated from a microorganism.
  • the transgenic DNA can be integrated into nuclear, mitochondrial or chloroplastic genomes through homologous or non-homologous recombination.
  • the transgenic DNA can also replicate without integrating into nuclear, mitochondrial or chloroplastic genomes, such as in an episomal vector.
  • the vector can contain a single, operably-linked set of regulatory elements that includes a promoter, a 5’ untranslated region (5’ UTR), an insertion site for transgenic DNA, a 3’ untranslated region (3’ UTR) and a terminator sequence.
  • Vectors useful in the present methods are well known in the art.
  • the term “episomal vector” refers to a DNA vector based on a bacterial episome that can be expressed in a transformed cell without integration into the transformed cell genome (Karas et al 2015).
  • Episomal vectors can be transferred from a bacteria (e,g, Escherichia coli) to another target microorganism (e.g. a microalgae) via conjugation.
  • the term “expression cassette” means a single, operably-linked set of regulatory elements that includes a promoter, a 5’ untranslated region (5’ UTR), an insertion site for transgenic DNA, a 3’ untranslated region (3’ UTR) and a terminator sequence.
  • the at least one nucleic acid molecule is an episomal vector.
  • operably-linked refers to an arrangement of two or more components, wherein the components so described are in a relationship permitting them to function in a coordinated manner.
  • a transcriptional regulatory sequence or a promoter is operably- linked to a coding sequence if the transcriptional regulatory sequence or promoter facilitates aspects of the transcription of the coding sequence.
  • the phrase “introducing a nucleic acid molecule into a microorganism” includes the stable integration of the nucleic acid molecule into the genome of a microorganism or the introduction of a replicating vector into a microorganism, or to the transient integration of the nucleic acid molecule into the microorganism.
  • the introduction of a nucleic acid into a cell is also known in the art as transformation.
  • the nucleic acid vectors may be introduced into the microorganism using techniques known in the art including, without limitation, agitation with glass beads, electroporation, agrobacterium- mediated transformation, an accelerated particle delivery method (i.e. particle bombardment), a cell fusion method or by any other method to deliver the nucleic acid molecule into a microorganism.
  • sequence identity refers to the percentage of sequence identity between two nucleic acid (polynucleotide) or two amino acid (polypeptide) sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • the determination of percent identity between two sequences can also be accomplished using a mathematical algorithm.
  • One non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990), modified as in Karlin and Altschul (1993).
  • Gapped BLAST can be utilized as described in Altschul et al. (1997).
  • PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Altschul et al., 1997).
  • the default parameters of the respective programs e.g., of XBLAST and NBLAST
  • Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988). Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • a PAM120 weight residue table When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
  • the nucleic acids are optimized for codon usage in a specific microalgal or cyanobacterial species.
  • the production of cannabinoid biosynthetic pathway products by a genetically engineered microorganism provided herein can be the result of introducing or increasing the activity of one or more enzymes associated with the cannabinoid biosynthetic pathway.
  • This can include, for example, the introduction of a nucleic acid molecule comprising a nucleic acid sequence encoding an enzyme of a cannabinoid biosynthetic pathway.
  • cannabinoid biosynthetic pathway enzymes include, but are not limited to hexanoyl-CoA synthetase, type III polyketide synthase (e.g., tetraketide synthase, Steely 1 and Steely 2), olivetolic acid cyclase, geranyl pyrophosphate synthase, aromatic prenyltransferase (APT), geranyl pyrophosphate:olivetolic acid geranyltransferasecannabichromene synthase, tetrahydrocannabinolic acid synthase (THCAS), and cannabidiolic acid synthase (CBDAS).
  • Amino acid sequences of cannabinoid biosynthetic pathway enzymes as described herein are provided in Table 1.
  • cannabinoid is generally understood to include any chemical compound that acts upon a cannabinoid receptor.
  • cannabinoids include cannabidiol (CBD), cannabinol (CBN), cannabigerol (CBG), cannabichromene (CBC), tetrahydrocannabivarin (THCV), cannabichromanon (CBCN), cannabielsoin (CBE), cannbifuran (CBF), tetrahydrocannabinol (THC), cannabinodiol (CBDL), cannabicyclol (CBL), cannabitriol (CBT), cannabivarin (CBV), cannabidivarin (CBDV), cannabichromevarin (CBCV), cannabigerovarin (CBGV), cannabigerol monomethyl ether (CBGM), cannabinerolic acid, cannabidiol (CBD), cannabinol (CBN), can
  • a cannabinoid may be in an acid form or a non-acid form, the latter also being referred to as the decarboxylated form since the non-acid form can be generated by decarboxylating the acid form.
  • a cannabinoid biosynthetic pathway product is a cannabinoid or a product associated with the production of a cannabinoid such as precursor or intermediate compound.
  • cannabinoid biosynthetic pathway products include, but are not limited to, hexanoyl-CoA, trioxododecanoyl-CoA, olivetolic acid, olivetol, divarinolic acid, divarinol, cannabigerolic acid, cannabigerol, A9-tetrahydrocanannabinolic acid, cannabidiolic acid, A9-tetrahydrocanannabinol and cannabidiol.
  • the cannabinoid biosynthetic pathway product is at least one of hexanoyl-CoA, trioxododecanoyl-CoA, olivetolic acid, olivetol, divarinolic acid, divarinol, cannabigerolic acid, cannabigerol, A9-tetrahydrocanannabinolic acid, cannabidiolic acid, D9- tetrahydrocanannabinol and cannabidiol.
  • Figure 1 shows an exemplary cannabinoid biosynthetic pathway based on enzymes from Cannabis sativa : Tetraketide synthase (TKS) condenses hexanoyl-CoA and malonyl-CoA to form the intermediate trioxododecanoyl-CoA; Olivetolic acid cyclase (OAC) catalyzes an intramolecular aldol condensation to yield olivetolic acid (OA); aromatic prenyltransferase transfers a geranyldiphosphate (GPP) onto OA to produce cannabigerolic acid (CBGA); tetrahydrocannabinolic acid synthase or cannabidiolic acid synthase catalyze the oxidative cyclization of CBGA into tetrahydrocannabinolic acid (THCA) or cannabidiolic acid (CBDA), respectively.
  • TTKS Tetraketide synthase
  • OAC cycl
  • biosynthetic intermediates can be used in a cannabinoid biosynthetic pathway in a genetically engineered microorganism.
  • olivetol is an intermediate that lacks the carboxyl group of olivetolic acid.
  • Use of olivetol instead of olivetolic acid in a cannabinoid biosynthetic pathway will produce cannabinoids that similarly lack a carboxyl group such as cannabigerol (CBG), tetrahydrocannabinol (THC), or cannabidiol (CBD).
  • CBD cannabigerol
  • THC tetrahydrocannabinol
  • CBD cannabidiol
  • tetraketide synthase condenses butyryl-CoA and malonyl-CoA to form the intermediate trioxodecanoyl-CoA
  • OAC olivetolic acid cyclase
  • Divarinolic acid is an intermediate containing an n-propyl group in place of the n-pentyl group found in olivetolic acid.
  • divarinolic acid instead of olivetolic acid in a cannabinoid biosynthetic pathway will produce cannabinoids that similarly contain an n-propyl group such as cannabigerovarinic acid (CBGVA), tetrahydrocannabivarinic acid (THCVA), cannabidivarinic acid (CBDVA), or cannabichromevarinic acid (CBCVA).
  • CBGVA cannabigerovarinic acid
  • THCVA tetrahydrocannabivarinic acid
  • CBDVA cannabidivarinic acid
  • CBCVA cannabichromevarinic acid
  • divarinol is an intermediate that lacks the carboxyl group of divarinolic acid, and contains an n-propyl group in place of the n-pentyl group found in olivetol.
  • cannabinoids that similarly contain an n-propyl group and lack a carboxyl group such as cannabigerovarin (CBGV), tetrahydrocannabivarin (THCV), cannabidivarinic acid (CBDV), or cannabichromevarinic acid (CBCV).
  • CBDGV cannabigerovarin
  • THCV tetrahydrocannabivarin
  • CBDV cannabidivarinic acid
  • CBCV cannabichromevarinic acid
  • alternative enzymes can be used in a cannabinoid biosynthetic pathway in a genetically engineered microorganism.
  • alternative enzymes of a cannabinoid biosynthetic pathway may be found in other plants (e.g., Humulus lupulus), in bacteria (e.g., Streptomyces), or in protists (e.g., Dictyostelium discoideum).
  • Enzymes that differ in structure, but perform the same function, may be used interchangeably in a cannabinoid biosynthetic pathway in a genetically engineered microorganism.
  • the aromatic prenyltransferases CsPT1 (SEQ ID NO: 3) and CsPT4 (SEQ ID NO: 10) from Cannabis sativa HIPT1 from Humulus lupulus (SEQ ID NO: 11), and Orf2 (SEQ ID NO: 9) from Streptomyces Sp.
  • Strain CI190 are all aromatic prenyltransferases that catalyze the synthesis of CBGA from GPP and OA.
  • the Steelyl (SEQ ID NO: 7) or Steely2 (SEQ ID NO: 8) polyketide synthase from Dictyostelium discoideum, or a variant thereof, can be used to condense malonyl-CoA into olivetol, and may be used in place of TKS to produce olivetol in the absence of OAC.
  • modified variants of these enzymes can be used in a cannabinoid biosynthetic pathway in a genetically engineered microorganism.
  • Variants of enzymes for use in a cannabinoid biosynthetic pathway can be generated by altering the nucleic acid sequence encoding said enzyme to, for example, increase/decrease the activity of a domain, add/remove a domain, add/remove a signaling sequences, or to otherwise alter the activity or specificity of the enzyme.
  • the sequence of Steelyl can be modified to reduce the activity of a methyltransferase domain in order to produce non-methylated cannabinoids.
  • this can be done by mutating amino acids G1516D+G1518A or G1516R relative to SEQ ID NO: 7 as disclosed in WO/2018/148849.
  • sequences of tetrahydrocannabinolic acid synthase or cannabidiolic acid synthase can be modified to remove an N-terminal secretion peptide.
  • this can be done by removing amino acids 1-28 of SEQ ID NO: 5 or 6 to produce a truncated enzyme as disclosed in WO/2018/200888.
  • a acyl-CoA synthetase is an acyl-activating enzyme that ligates CoA and a straight-chain alkanoic acid or alkanoate containing 2 to 6 carbon atoms to produce alkanoyl-CoA, wherein the alkanoyl-CoA is a thioester of coenzyme A containing an alkanoyl group of 2 to 6 carbon atoms.
  • the acyl-CoA synthetase is hexanoyl-CoA synthetase, which ligates CoA and hexanoic acid or hexanoate to produce hexanoyl-CoA.
  • a hexanoyl-CoA synthetase may have the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence with at least 90% identity to SEQ ID NO: 4.
  • an acyl-CoA synthetase ligates CoA and butyric acid or butyrate to produce butyryl-CoA.
  • a type III polyketide synthase is an enzyme that produces polyketides by catalyzing the condensation reaction of acetyl units to thioester-linked starter molecules.
  • a type III polyketide synthase may have the amino acid sequence of SEQ ID NO: 1 , 7, or
  • a type III polyketide synthase condenses an alkanoyl-CoA with three malonyl-CoA in a multi-step reaction to form a 3,5,7-trioxoalkanoyl-CoA, wherein the 3,5,7-trioxoalkanoyl-CoA contains 8 to 12 carbon atoms.
  • the type III polyketide synthase is tetraketide synthase from Cannabis sativa which is also known in the art as olivetol synthase and 3,5,7-trioxododecanoyl-CoA synthase.
  • tetraketide synthase condenses hexanoyl-CoA with three malonyl-CoA in a multi-step reaction to form 3,5,7-trioxododecanoyl-CoA.
  • tetraketide synthase condenses butyryl-CoA with three malonyl-CoA in a multi-step reaction to form 3,5,7-trioxodecanoyl-CoA.
  • the type III polyketide synthase is Steelyl or Steely 2 from Dictyostelium discoideum, comprising a domain with type III polyketide synthase activity, ora variant thereof (e.g., Steelyl (G1516D+G1518A) or Steelyl (G1516R) disclosed in WO/2018/148849).
  • Steelyl is also known in the art as DiPKS or DiPKSI
  • Steely2 is also known in the art as DiPKS37.
  • An olivetolic acid cyclase refers to an enzyme that catalyzes an intramolecular aldol condensation of a 3,5,7-trioxoalkanoyl-CoA to form a 2,4-dihydroxy-6-alkylbenzoic acid, wherein the alkyl group of the benzoic acid contains 1 to 5 carbons.
  • an olivetolic acid cyclase catalyzes the formation of olivetolic acid from 3,5,7-trioxododecanoyl-CoA.
  • an olivetolic acid cyclase catalyzes the formation of divarinolic acid from 3,5,7-trioxodecanoyl-CoA.
  • An olivetolic acid cyclase may have the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence with at least 90% identity to SEQ ID NO: 2.
  • Olivetolic acid cyclase from Cannabis sativa is also known in the art as olivetolic acid synthase and 3,5,7- trioxododecanoyl-CoA CoA-lyase.
  • An aromatic prenyltransferase refers to an enzyme capable of transferring a geranyl diphosphate onto a 5-alkylbenzene-1 ,3-diol to synthesize a 2- geranyl-5-alkylbenzene-1 ,3-diol, wherein the alkyl group of the product contains 1 to 5 carbons.
  • an aromatic prenyltransferase transfers a geranyl disphosphate onto olivetol to synthesize cannabigerol (CBG).
  • an aromatic prenyltransferase transfers a geranyl disphosphate onto olivetolic acid (OA) to synthesize cannabigerolic acid (CBGA).
  • an aromatic prenyltransferase transfers a geranyl disphosphate onto divarinolic acid to synthesize cannabigerovarin (CBGV).
  • an aromatic prenyltransferase transfers a geranyl disphosphate onto divarinolic acid to synthesize cannabigerovarinic acid (CBGVA).
  • aromatic prenyltransferase is aromatic prenyltransferase from Cannabis sativa which is also known in the art as CsPT1 , prenyltransferase 1 , geranylpyrophosphate-olivetolic acid geranyltransferase, and geranyl-diphosphate: olivetolate geranytransferase.
  • aromatic prenyltransferase include HIPT1 from Humulus lupulus , CsPT4 from Cannabis sativa, and Orf2 (NphB).
  • Orf2 enzyme can be wild type Orf2 (e.g. from Streptomyces Sp.
  • Strain CI190 or a mutant Orf2 enzyme engineered to produce a higher amount of enzymatic product and/or to to have higher activity in relation to a specific substrate.
  • mutant Orf2 enzymes are disclosed in WO2019/183152A1 , the contents of which are herein incorporated by reference.
  • An aromatic prenyltransferase may have the amino acid sequence of SEQ ID NO: 3, 9, 10 or 11 , or an amino acid sequence with at least 90% identity to SEQ ID NO: 3, 9, 10 or 11 .
  • a tetrahydrocannabinolic acid synthase is also known in the art as D9- tetrahydrocannabinolic acid synthase, and synthesizes A9-tetrahydrocannabinolic acid by catalyzing the cyclization of the monoterpene moiety in cannabigerolic acid.
  • a tetrahydrocannabinolic acid synthase may have the amino acid sequence of SEQ ID NO:
  • a cannabidiolic acid synthase synthesizes cannabidiolic acid by catalyzing the stereoselective oxidative cyclization of the monoterpene moiety in cannabigerolic acid.
  • a cannabidiolic acid synthase may have the amino acid sequence of SEQ ID NO:
  • genetically modified microorganisms comprise at least one nucleic acid molecule that encodes at least one, two, three, four, five, or six of hexanoyl-CoA synthetase, type III polyketide synthase (e.g., tetraketide synthase, Steelyl and Steely2), olivetolic acid cyclase, aromatic prenyltransferase, tetrahydrocannabinolic acid synthase, or cannabidiolic acid synthase.
  • type III polyketide synthase e.g., tetraketide synthase, Steelyl and Steely2
  • olivetolic acid cyclase e.g., aromatic prenyltransferase, tetrahydrocannabinolic acid synthase, or cannabidiolic acid synthase.
  • the at least nucleic acid molecule encodes at least one of a hexanoyl-CoA synthetase comprising an amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO: 4; a type III polyketide synthase comprising an amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO: 1 , 7 or 8; an olivetolic acid cyclase comprising an amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO: 2 or 17; an aromatic prenyltransferase comprising an amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO: 3, 9, 10, or 11
  • microorganisms for use in the present invention can be microorganisms capable of producing at least one cannabinoid biosynthetic pathway product.
  • the microorganism capable of producing at least one cannabinoid biosynthetic pathway product is cultured in a culture medium comprising a precursor or intermediate compound of the cannabinoid biosynthetic pathway.
  • the microorganism may convert the precursor of intermediate compound into another cannabinoid biosynthetic pathway product.
  • the microorganism capable of producing at least one cannabinoid biosynthetic pathway product is cultured in a culture medium comprising a 2,4-dihydroxy- 6-alkylbenzoic acid or a 2,4-dihydroxy-6-alkylbenzoate.
  • the microalga is from the genera Ankistrodesmus, Asteromonas, Auxenochlorella, Basichlamys, Botryococcus, Botryokoryne, Borodinella, Brachiomonas, Catena, Carteria, Chaetophora, Characiochloris, Characiosiphon, Chlainomonas, Chlamydomonas, Chlorella, Chlorochytrium, Chlorococcum, Chlorogonium, Chloromonas, Closteriopsis, Dictyochloropsis, Dunaliella, Ellipsoidon, Eremosphaera, Eudorina, Floydiella, Friedmania, Haematococcus, Hafniomonas, Heterochlorella, Gonium, Halosarcinochlamys, Koliella, Lobocharacium, Lobochlamys, Lobomonas, Lobosphaera, Lobosphaeropsis
  • the microalga is Chlamydomonas reinhardtii, Chlorella vulgaris, Chlorella sorokiniana, Chlorella protothecoides, Tetraselmis chui, Nannochloropsis oculate, Scenedesmus obliquus, Acutodesmus dimorphus, Dunaliella tertiolecta , or Heamatococus plucialis.
  • the microalga is a diatom, optionally Phaeodactylum tricornutum or Thalassiosira pseudonana.
  • the cyanobacterium is from Spirulinaceae, Phormidiaceae, Synechococcaceae, or Nostocaceae.
  • the cyanobacterium is Arthrospira plantesis , Arthrospira maxima, Synechococcus elongatus, or Aphanizomenon flos-aquae.
  • Microorganisms can be grown in organic conditions without the use of chemicals or additives that contravene the standards for organically-produced products.
  • Microalgae can be grown organically, for example, by growing them in conditions that comply with jurisdictional standards such as the standards set by the United States (US Organic Food Production Act; USDA National Organic Program Certification; USDA Organic Regulations), the European Union (Regulation No 834/2007 prior to January 1 , 2021 ; Regulation 2018/848 from January 1 , 2021), and Canada (Canadian Food Inspection Agency Canadian Organic Standards).
  • US Organic Food Production Act US Organic Food Production Act
  • USDA National Organic Program Certification USDA Organic Regulations
  • the European Union Regulation No 834/2007 prior to January 1 , 2021
  • Regulation 2018/848 from January 1 , 2021 Regulation 2018/848 from January 1 , 2021
  • Canada Canadian Food Inspection Agency Canadian Organic Standards
  • a method of culturing a photosynthetic microorganism comprising: a. culturing the photosynthetic microorganism under a first light spectrum for a first period of time; and b. culturing the photosynthetic microorganism under a second light spectrum for a second period of time, wherein the second light spectrum is different from the first light spectrum.
  • the photosynthetic microorganism comprises at least one nucleic acid molecule encoding at least one cannabinoid biosynthetic pathway enzyme.
  • the at least one nucleic acid molecule encodes at least one of a hexanoyl-CoA synthetase, a type III polyketide synthase (e.g., tetraketide synthase, Steely 1 and Steely 2), an olivetolic acid cyclase, an aromatic prenyltransferase (e.g. CsPT1 , Orf2, CsPT4, and HIPT1), a tetrahydrocannabinolic acid synthase, or a cannabidiolic acid synthase.
  • a hexanoyl-CoA synthetase e.g., tetraketide synthase, Steely 1 and Steely 2
  • an olivetolic acid cyclase e.g.,
  • hexanoyl-CoA synthetase comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO: 4.
  • the olivetolic acid cyclase comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO: 2.
  • aromatic prenyltransferase comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO: 3, 9, 10, or 11.
  • tetrahydrocannabinolic acid synthase comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO: 5.
  • cannabidiolic acid synthase comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the sequence as shown in SEQ ID NO: 6.
  • the at least one cannabinoid biosynthetic pathway product is at least one of hexanoyl-CoA, trioxododecanoyl-CoA, olivetolic acid, olivetol, cannabigerolic acid, cannabigerol, D9- tetrahydrocanannabinolic acid, cannabidiolic acid, A9-tetrahydrocanannabinol and cannabidiol.
  • the first light spectrum comprises at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of light within about 590 nm to about 740 nm in wavelength; or comprises about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% of light within about 590 nm to about 740 nm in wavelength; or comprises about 65% to about 95%, about 70% to about 95%, about 75% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 65% to about 90%, about 70% to about 90%, about 75% to about 90%, about 80% to about 90%, about 85% to about 90%, about 65% to about 85%, about 70% to about 85%, about 75% to about 85%, about 80% to about 85%, about 65% to about 80%, about 70%
  • the first light spectrum comprises at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of light within about 625 nm to about 740 nm in wavelength; or comprises about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% of light within about 590 nm to about 740 nm in wavelength; or comprises about 65% to about 95%, about 70% to about 95%, about 75% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 65% to about 90%, about 70% to about 90%, about 75% to about 90%, about 80% to about 90%, about 85% to about 90%, about 65% to about 85%, about 70% to about 85%, about 75% to about 85%, about 80% to about 85%, about 65% to about 80%, about 70%
  • the first light spectrum comprises at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of light within about 600 nm to about 700 nm in wavelength; or comprises about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% of light within about 590 nm to about 740 nm in wavelength; or comprises about 65% to about 95%, about 70% to about 95%, about 75% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 65% to about 90%, about 70% to about 90%, about 75% to about 90%, about 80% to about 90%, about 85% to about 90%, about 65% to about 85%, about 70% to about 85%, about 75% to about 85%, about 80% to about 85%, about 65% to about 80%, about 70% to about
  • the first light spectrum comprises at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of light within about 600 nm to about 640 nm; or comprises about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% of light within about 590 nm to about 740 nm in wavelength; or comprises about 65% to about 95%, about 70% to about 95%, about 75% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 65% to about 90%, about 70% to about 90%, about 75% to about 90%, about 80% to about 90%, about 85% to about 90%, about 65% to about 85%, about 70% to about 85%, about 75% to about 85%, about 80% to about 85%, about 65% to about 80%, about 70% to about
  • the first light spectrum comprises at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% of light within about 680 nm to about 700 nm; or comprises about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% of light within about 590 nm to about 740 nm in wavelength; or comprises about 65% to about 95%, about 70% to about 95%, about 75% to about 95%, about 80% to about 95%, about 85% to about 95%, about 90% to about 95%, about 65% to about 90%, about 70% to about 90%, about 75% to about 90%, about 80% to about 90%, about 85% to about 90%, about 65% to about 85%, about 70% to about 85%, about 75% to about 85%, about 80% to about 85%, about 65% to about 80%, about 70% to about
  • a method of culturing a photosynthetic microorganism capable of producing at least one cannabinoid biosynthetic pathway product in a culture medium comprising: a. culturing the photosynthetic microorganism under a first light spectrum for a first period of time; and b. culturing the photosynthetic microorganism under a second light spectrum for a second period of time, wherein the second light spectrum is different from the first light spectrum, and wherein the culture medium comprises a 2,4-dihydroxy-6-alkylbenzoic acid or a 2,4-dihydroxy-6- alkylbenzoate.
  • the at least one cannabinoid biosynthetic pathway product is at least one of cannabigerolic acid, cannabigerol, D9- tetrahydrocanannabinolic acid, cannabidiolic acid, A9-tetrahydrocanannabinol, and cannabidiol.
  • the first light spectrum comprises at least 65% of light within about 600 nm to about 640 nm in wavelength.
  • the first light spectrum comprises at least 65% of light within about 680 nm to about 700 nm in wavelength.
  • Phaetodactylum Tricornutum (Culture Collection of Algae and Protozoa CCAP 1055/1) were grown in L1 Media without silica (Artificial Sea Water) in 250 mL Erlenmeyer flasks (50 mL culture volume) for analysis of biomass and lipids or in flat-bottom 6 well culture plates (5 mL culture colume) for analysis of cell growth.
  • P.tricornutum cultures were grown at 18 ⁇ 1 °C and maintained under a continuous light- dark cycle of 16/8 hours using white LED light (F54T5/841) kept 60cm above the bottom of the culture flask.
  • Coloured light (red light and yellow light) was provided by covering the flasks with coloured cellophane. Experiments were conducted in a CMP6050 chamber with a light intensity of 75 pMOL nr 2 s _1 , humidity of 50%, and shaker speed of 130 rpm.
  • the first set of experiments examined the impact of red light (RL), yellow light (YL) and white light (WL) in both mixotrophic and autotrophic conditions under growth conditions explained above.
  • RL red light
  • YL yellow light
  • WL white light
  • P.tricornutum was cultivated in mixotrophic conditions and autotrophic conditions in the three different light conditions (Table 2).
  • the cultures were subjected to consistent shaking in orbital shaker at 130 rpm. Cell growth was monitored by measuring the optical density at 680 nm by Synergy Microplate Reader, BioteK on every second day. All the conditions were studied in triplicates. [0082] The biomass studied for the initial conditions was collected after centrifugation of algal cultures at 7000 g for 10 min on the 10th day. The supernatant was discarded and pellet was dried at 60 °C for biomass analysis by measurement on a scale.. The selected conditions were studied for further experimentation.
  • Intracellular lipid bodies were visualized via modified Nile Red (9- diethylamino-5H-benzo[a]-phenoxazine-5-one) staining. Briefly, 1 mL of the algal culture was centrifuged at 12,000 rpm for 10 minutes. The pellet was resuspended in 500 uL of 20% Dimethyl sulfoxide (DMSO) and vortexed for 1 minute at room temperature. Cells were centrifuged at 12,000 rpm for 5 minutes. The pellet was suspended in 500 uL of water and vortexed before adding Nile Red (250 uL of 0.5 mg/mL dissolved in acetone) and incubated for 5 minutes in the dark at room temperature. Stained cells were visualized under a fluorescent microscope using UV light with excitation and emission at 485 nm and 552 nm, respectively.
  • modified Nile Red 9- diethylamino-5H-benzo[a]-phenoxazine-5-one
  • Total lipids were extracted using Bligh and Dyer method (Bligh and Dyer 1959) with some modifications.
  • Bligh and Dyer method Bligh and Dyer 1959
  • Nile red assay was performed on 96 well plates containing 250 uL of sample with 15 uL of Nile red dissolved in acetone from the stock solution of 0.5 mg/mL. The fluorescence intensity was measured at excitation of 530 nm and emission of 590 nm using Synergy Microplate Reader, BioteK.
  • Lipids were extracted for the preparation of FA methyl esters (FAMEs) following standard protocols as previously described (Budge et al 2006). Each sample was homogenized, and lipids were extracted using a mixture of chloroform/methanol (2:1). FAMEs were prepared through acidic transesterification using sulfuric acid in methanol and quantified using temperature-programmed gas liquid chromatography on a Perkin Elmer Autosystem II Capillary FID gas chromatograph fitted with a 30 m x 0.25 mm FID column coated with 50 % cyanopropyl-methylpolysiloxane (DB-23) and linked to a computerized integration system (Varian Star software).
  • FAMEs FA methyl esters
  • the fatty acid methyl ester analysis revealed that: saturated fatty acids (SFA) as a percent of total lipids was 24% in red light (R) and red light shifting to white light (red light during lag and exponential phases and white light in stationary phase, RS) and 26% in white light (C); monounsaturated fatty acids (MUFA) as a percent of total lipids was comparable in C and R conditions at 31% but was lower in RS conditions at 28%; and polyunsaturated fatty acids as a percent of total lipids was 27%, 26%, and 28% in C, R, and RS conditions, respectively. The remaining fraction of total lipids was composed of non-neutral lipids. Individual fatty acid species and their quantity as a percent of total lipids in each condition is shown in Table 5:
  • the R condition also decreased the percent quantity of myristic acid (C14:0), a medium-chain fatty acid, compared to the C condition, but the percent quantity of this fatty acid is recovered in the RS condition.
  • Medium-chain fatty acids i.e. , fatty acids containing between 6 and 14 carbons (C:6 - C: 14)
  • C:6 - C: 14 may improve the solubility of cannabinoids in the lipid bodies of P.tricornutum.
  • the properties and structural features (chain length, unsaturation and branching) of fatty acids determine the properties of the total fatty acid fraction.
  • the overall fatty acid composition was analyzed using Biodiesel Analyzer (Talebi et al 2014) and parameters of the fatty acid fraction from the different conditions were compared to those of Biodiesel standards EN 14214:2008 and ASTM D6751 (Table 6).
  • Figure 9 shows the transmittance spectrum of the red light filter used to provide the red light for conditions (r) and (rs). As shown in Figure 10, switching to white light at the stationary phase after culturing cells under blue light (Bs) or yellow light (Ys) during the lag and exponential phases did not improve biomass yield, whereas an improvement was seen in cells cultured under red light (rs) before switching to white light at the stationary phase.
  • Bs blue light
  • Ys yellow light
  • the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps.
  • the foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives.
  • the term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but exclude the presence of other unstated features, elements, components, groups, integers and/or steps.

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Abstract

L'invention concerne également un procédé de culture d'un micro-organisme photosynthétique comprenant la culture du micro-organisme photosynthétique dans un premier spectre de lumière pendant une première période de temps et dans un second spectre de lumière pendant une seconde période de temps. L'invention concerne en outre un procédé de culture d'un micro-organisme photosynthétique capable de produire au moins un produit de biosynthèse dans un milieu de culture comprenant la culture du micro-organisme photosynthétique dans un premier spectre de lumière pendant une première période de temps et dans un second spectre de lumière pendant une seconde période de temps, le milieu de culture comprenant un acide 2,4-dihydroxy-6-alkylbenzoïque ou un 2,4-dihydroxy-6-alkylbenzoate.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11274320B2 (en) 2019-02-25 2022-03-15 Ginkgo Bioworks, Inc. Biosynthesis of cannabinoids and cannabinoid precursors

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2740349A1 (fr) * 2011-08-05 2014-06-11 Showa Denko K.K. Procédé de culture d'algues et équipement de culture d'algues
US20190194598A1 (en) * 2016-09-01 2019-06-27 Showa Denko K.K. Method for culturing photosynthetic microalgae
US20190223397A1 (en) * 2016-09-01 2019-07-25 Showa Denko K.K. Method for culturing photosynthetic microalgae
WO2019210404A1 (fr) * 2018-04-30 2019-11-07 Algae-C Inc. Micro-organisme modifié pour la production de produits de la voie de biosynthèse des cannabinoïdes

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2740349A1 (fr) * 2011-08-05 2014-06-11 Showa Denko K.K. Procédé de culture d'algues et équipement de culture d'algues
US20190194598A1 (en) * 2016-09-01 2019-06-27 Showa Denko K.K. Method for culturing photosynthetic microalgae
US20190223397A1 (en) * 2016-09-01 2019-07-25 Showa Denko K.K. Method for culturing photosynthetic microalgae
WO2019210404A1 (fr) * 2018-04-30 2019-11-07 Algae-C Inc. Micro-organisme modifié pour la production de produits de la voie de biosynthèse des cannabinoïdes

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
D'ADAMO ET AL.: "Engineering the unicellular alga Phaeodactylum tricornutum for high- value plant triterpenoidproduction", PLANT BIOTECHNOL J, vol. 17, no. 1, 19 June 2018 (2018-06-19), pages 75 - 87, XP055822420, ISSN: 1467-7644, DOI: https://dx.doi.org/10.1111%2Fpbi. 12948 *
SHARMA ET AL.: "Red Light Variation an Effective Alternative to Regulate Biomass and Lipid Profiles in Phaeodactylum tricornutum", APPL SCI, vol. 10, no. 7, 7 April 2020 (2020-04-07), pages 1 - 18, XP055822426, ISSN: 2076-3417, DOI: https://doi.org/10.3390/appl0072531 *
SUNG ET AL.: "Wavelength shift strategy to enhance lipid productivity of Nannochloropsis gaditana", BIOTECHNOL BIOFUELS, vol. 11, no. 70, 19 March 2018 (2018-03-19), pages 1 - 12, XP055822415, ISSN: 1754- 6834, DOI: https://doi.org/l0.1186/s13068-018-1067-2 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11274320B2 (en) 2019-02-25 2022-03-15 Ginkgo Bioworks, Inc. Biosynthesis of cannabinoids and cannabinoid precursors

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